Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

Introduction Saccharomyces cerevisiae is a yeast widely used for commercial fermentation to create diverse foodstuffs, including bread, beer, wine, coffee, and chocolate. It is also an important model organism for studying fundamental principles of metabolism and eukaryotic cell biology, including host-virus interactions. Like many fungi, S. cerevisiae harbors different parasitic genetic elements, including RNA viruses, DNA plasmids, and retrotransposons. These RNA viruses have no known extracellular stage in their lifecycle and transmit vertically during cell division or horizontally by mating [1,2]. Infection of fungi by viruses (mycoviruses) often does not result in overt signs of disease, but there are examples of how mycoviruses can influence fungal physiology [3][4][5][6][7][8][9].
The discovery of the first mycovirus in S. cerevisiae was due to the production of antifungal toxins, a phenotype often dependent on double-stranded RNA (dsRNA) viruses from the family Totiviridae and toxin-encoding satellite dsRNAs [10][11][12]. Single-stranded RNA viruses of S. cerevisiae (from the family Narnaviridae) were discovered by the detection of dsRNA replication intermediates [13]. Contemporary RNA sequencing techniques have enabled the discovery of many more mycoviruses, either from individual strains of fungi or from environmental samples, including novel viruses from the Totiviridae and Narnaviridae virus families in yeasts of the Saccharomycotina subphylum [14][15][16][17][18]. Due to its short generation time, ease of genetic manipulation, and the large array of genetic tools and other resources, S. cerevisiae is an ideal model organism for studying viruses and other parasitic genetic elements. Such studies have revealed mechanistic details relating to virus replication, persistence, and inheritance, as well as the cellular mechanisms that suppress viral replication [1,2].
Partitiviruses (PVs) have been found within the Basidiomycota and Ascomycota fungi, as well as plants and protozoa [19]. PVs are also associated with invertebrates and can actively replicate in insect cells and tissues, with some phenotypic changes to infected insects [20][21][22]. PV genomes comprise of two linear dsRNA molecules that range in size between 1.4 and 2.4 kbp. Each genome segment is separately packaged into a viral particle (i.e. biparticulate) [23]. One genome segment encodes the RNA-dependent RNA polymerase (RdRP) responsible for genome replication and transcription of viral RNAs. The second segment encodes the coat protein (CP) that encapsidates the viral genome [24]. Assembled PV virions typically measure 25-40 nm in diameter and have isometric T = 1 symmetry constructed of 60 homodimeric subunits per unenveloped particle. Five PV particle structures have been obtained, and all contain a striking protrusion domain (P-domain) that projects from the surface of the viral shell domain (S-domain) and forms intermolecular interactions between CP monomers [25][26][27][28][29]. PV particles can encapsidate two copies of each genome segment, and RNA transcripts are thought to be extruded from the virions into the cytoplasm for translation [30]. There are currently five approved genera within the family Partitiviridae, named Alphapartitivirus, Betapartitivirus, Gammapartitivirus, Deltapartitivirus, and Cryspovirus, based on criteria such as genome length, host species, and the relatedness of the RdRP and CP genes ( [19,31] and International Committee on Taxonomy of Viruses (ICTV): https://ictv.global/taxonomy/). The new genera Epsilonpartitivirus and Zetapartitivirus were recently proposed based on the discovery of several novel viruses [32,33]. PVs that persistently infect fungi have been found within all PV genera except Deltapartitivirus and Cryspovirus, which infect plants and protozoa, respectively.
PV infection was long considered cryptic, with few consequences for the infected cell. However, in fungi, infection by PVs can result in a slower growth rate, reduced conidia formation, altered morphology, and the disruption of sexual reproduction [34][35][36][37][38]. This can lead to hypovirulence in fungal pathogens relevant to agriculture [39][40][41] but not in all cases [33,35]. Of relevance to human health, PV infection can cause hypervirulence in the pathogenic fungus Talaromyces marneffei [42] and has been suggested to increase the fecundity of the pathogenic protozoan Cryptosporidium parvum [43]. The discovery of PVs in S. cerevisiae provides a unique opportunity to leverage the powerful genetic tools of this model organism to better understand their replication and effect on host physiology.

Double-stranded RNA partitiviruses identified in S. cerevisiae
To take an unbiased approach to understanding virus diversity in S. cerevisiae, dsRNAs were purified from a random selection of 520 strains from the 1,002 Yeast Genomes Project collection (S1 Table) [44]. These dsRNAs were analyzed by a short-read sequencing pipeline that identified 110 contigs with significant sequence similarity to known virus families (Fig 1A and  S2 Table) [45,46]. Contigs with sequences similar to the Totiviridae were most frequently DsRNAs extracted in panel B were subjected to incubation at 37˚C with and without RNase III, and the resulting products were analyzed by agarose gel electrophoresis. (D) Two-step RT-PCR of total nucleic acids extracted from S. cerevisiae targeting the CP or RdRP genes with (+RT) or without (-RT) reverse transcriptase. Strain BJH001 was used as a negative control as it does not harbor PVs. Primers identified. Contigs with similarity to satellite dsRNAs were also identified with ORFs corresponding to the S. cerevisiae killer toxins K1, K2, Klus, and S. paradoxus killer toxins K66 and K21. After totiviruses and satellites, the next highest coverage score was for viruses of the Partitiviridae [19]. Other virus families identified by these analyses were represented by three or fewer contigs and were not investigated further (S2 Table). Assembled contigs identified sequences similar to previously identified virus proteins in Saccharomyces yeasts that were not fully characterized [47,48].
To confirm the presence of PVs in S. cerevisiae, 161 strains from the 1,002 Yeast Genomes Project collection, 146 strains from the collection of Ludlow et al., and three additional strains from other culture collections were screened using cellulose chromatography to extract dsRNAs (S3 Table) [44,49]. This approach identified PVs by the characteristic electrophoretic mobility of their dsRNAs, with 14% of strains containing PVs, mainly from the collection of Ludlow et al (Table 1). For example, purifying dsRNAs from S. cerevisiae strains YO858 and Y-5509 identified three distinct nucleic acid molecules with similar electrophoretic mobilities in both strains (Fig 1B). The RNAs were confirmed to be dsRNAs by RNase III digestion ( Fig  1C). These dsRNAs likely represent a monopartite totivirus genome (�4.6 kbp) and the bipartite genome of a PV (�1.5 kbp) encoding an RdRP (dsRNA1) and CP (dsRNA2). Finally, reverse transcriptase-PCR (RT-PCR) confirmed PVs in total nucleic acid extracts of S. cerevisiae strains YO858 and Y-5509 ( Fig 1D). PCR alone did not result in the amplification of PV sequences, and all samples were positive for the yeast chromosomal gene UBC6 (Fig 1D). S. cerevisiae BJH001 was included as a negative control as it is not infected with PVs [45].
In Saccharomyces yeasts, dsRNAs smaller than 4 kbp have been associated with non-autonomously replicating satellites [1,2]. These satellites are often responsible for encoding antifungal killer toxins maintained by the replicative machinery of totiviruses. One strain of killer yeast (S. cerevisiae CYC1172 (Fig 2A)) was discovered to harbor dsRNAs corresponding to a totivirus and three species of low molecular weight dsRNAs ( Fig 2B). This suggested the coexistence of a totivirus, PV, and a satellite dsRNA. To confirm that one of the three low molecular weight dsRNAs was a satellite, strain CYC1172 was grown on agar containing cycloheximide. This resulted in the curing of the satellite dsRNA and the loss of the killer phenotype, but not the curing of the totivirus or PV (Fig 2A and 2B). Moreover, screens of S. cerevisiae strains for dsRNAs identified 15 strains with PVs that lacked totiviruses (4.8% of all yeasts screened; Table 1), supporting the autonomy of PVs ( Fig 2C).

PVs from S. cerevisiae are most closely related to Cryptosporidium parvum virus 1
ScPVs were significantly enriched in strains isolated from coffee (25/44, Fisher's exact test p<0.01) and cacao (16/44, Fisher's exact test p<0.01) (Fig 3A) (S3 Table). Only three strains of S. cerevisiae with PVs (Y-5509, CYC1172, and ICV D254 Lalvin) were isolated from other environments (coconut pods and grape must). To characterize the genome sequence and organization of PVs from S. cerevisiae (ScPVs), complete ORFs were assembled for 14 ScPVs that corresponded to both an RdRP (486-491 amino acids) and a CP (280-310 amino acids) using short read sequencing (S1 Fig and S4 Table). ScPV proteins were smaller than PVs from plants, fungi, and protozoa, except for the RdRPs from the Deltapartitivirus genus ( Fig 3B). An alignment of the RdRP from 14 ScPVs and 45 representative sequences from five official and two unofficial Partitiviridae genera was constructed for phylogenetic analysis using maximum likelihood. All ScPVs clustered as a single monophyletic group, with CSpV1 from the protozoan C. parvum as the most closely related outgroup ( Fig 3C). The percent amino acid identity between ScPV and CSpV1 RdRPs was between 33-38%, and the CPs between 17-23% (S5 Table). The clustering of ScPVs suggests that there are three distinct species, which have been named Saccharomyces cerevisiae partitivirus-1, -2, and -3, represented by ScPVs isolated from strains Y-5509 (ScPV1-5509), YO858 (ScPV2-858), and CYC1172 (ScPV3-1172), respectively. The amino acid identities of the RdRP or CP from these three species are less than 90% identical, supporting their categorization as distinct species (S5 Table). The partial sequence of an ScPV (named ScCV1) was previously identified in a European oenological yeast strain [48], which has 98.78% amino acid sequence identity to ScPV3-1172, isolated from a European wine strain (La Rioja, Spain) in grape must.
Having identified many strains of S. cerevisiae with ScPVs, RT-PCR, and short-read sequencing were used to determine which species of ScPV were present in each yeast strain (S6 Table and Table 1). Eight strains harbored ScPV1, 16 strains ScPV2, and 16 strains were co-infected with ScPV1 and ScPV2. ScPV3 was only found in two European wine strains. Coinfection of strains with ScPV1 and ScPV2 was confirmed using gel electrophoresis by the resolution of doublets of dsRNA1 (S2 Fig).
The complete genome sequences of ScPV1-5509, ScPV2-858, and ScPV3-1172 were determined by 5' rapid amplification of cDNA ends (RACE) (Fig 4A). Overall, the bipartite organization of ScPVs was the same as CSpV1, with similar length genome segments and untranslated regions (UTRs). Analysis of the 5' UTRs of these viruses by mFold revealed commonalities in RNA secondary structure between genome segments of each ScPV and extensive structure in dsRNA2 ( Fig 4A and S3 Fig). The sequence analysis of the 3' UTRs of ScPVs identified conserved sequence elements (CSEs) in dsRNA1 and dsRNA2 that were unique to each   [50]. The RdRP from the picobirnavirus OaPV was used as an outgroup. The VT amino-acid exchange rate matrices were selected with a gamma distribution (G), a proportion of invariable sites (I), and empirical amino acid frequencies in the alignment fit these data best, as judged by PROTTEST [51]. The numbers at each node are the bootstrap values from 1000 iterations. The scale bar represents the distance of one amino acid substitution per site. The amino acid sequences in the phylogeny are from the viruses listed in S7 Table. https://doi.org/10.1371/journal.ppat.1011418.g003

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae virus species, including CSpV1 ( Fig 4B) [52]. The CSEs of ScPV1-5509 and ScPV2-858 were 32 bp and 40 bp, and 100% identical when comparing the UTRs of dsRNA1 and dsRNA2. ScPV3-1172 encoded a CSE of 36 bp but with 84% identity. There was no homology between CSEs from different ScPV species, and all were predicted to form small stem-loop structures that could be involved in viral RNA replication or packaging.

ScPVs assemble spherical particles with structural similarity to the PV shell domain
Previously described PVs formed spherical particles that package viral RNAs for replication and transcription. To determine whether ScPVs assemble similar particles, a strain of S. cerevisiae only infected with ScPVs was used for viral particle purification. Spherical virus-like particles with a diameter of �30 nm that lacked obvious P-domain surface protrusions were observed by TEM after sucrose gradient fractionation ( Fig 5A).
To determine the tertiary structure of the CP monomer of ScPVs, ab initio models were generated using AlphaFold2. The N-terminus of all ScPV CPs and the Cryspovirus CSpV1 were modeled with confidence of up to 80% that sharply declined after �150 amino acids (S4  Table). The Nterminal domain of the ScPV CP was mostly α-helical and with a long α3-helix cradled by four shorter α-helices, (α2, α4, α5, and α6) forming a rhomboid shell domain (S-domain) prototypical of PVs ( Fig 5B). DALI analysis of the ScPV CP revealed structural similarity to the CP of pepper cryptic virus 1 (PCV1; PDB:7ncr) with an average root mean squared deviation (RMSD) of 4.34 Å (Fig 5C). Superimposition of the ScPV CP in the context of a virus particle from PCV1 showed that the ScPV S-domains likely form the majority of the particle structure

The RdRPs of ScPVs share structural homology with the polymerases of the Picornaviridae
The same molecular modeling approach as performed with the ScPV CPs was also applied to predict the tertiary structure of the RdRPs of ScPVs and CSpV1, with all models having 95% confidence across the entire sequence (  Table). The tertiary structure organization of the modeled structures included the characteristic fingers, palm, and thumb domains, with a tertiary structure similar to human poliovirus RdRP (PDB:2ijd-1 and PDB:1ra6, average RMSD <3.1 Å) ( Fig 5D). As expected, motifs A-F aligned well between ScPVs, CSpV1, and poliovirus ( Fig 5E and S7 Fig). The close similarity of these RdRPs supports the common ancestry of the Partitiviridae and the Picornaviridae and their membership within the phylum Pisuviricota.

ScPVs are maintained in S. cerevisiae under laboratory conditions and inherited in a non-mendelian manner after sporulation
To leverage the power of S. cerevisiae as a model system to study PVs, it was important to determine whether these viruses are stably maintained under laboratory conditions. Four strains of S. cerevisiae containing ScPVs were maintained on agar plates incubated at either room temperature or 4˚C for 6 weeks. The presence of ScPVs was monitored by the extraction of dsRNAs and indicated that ScPVs were stably maintained (S8 Fig). PVs have been shown to transmit inefficiently during spore formation in certain fungal species [31], which would limit genetic approaches to studying virus-host interactions in S. cerevisiae. To determine whether ScPVs can segregate efficiently to daughter cells during meiosis, sporulation was induced in two diploid strains infected with ScPVs. Total nucleic acids were extracted from each haploid progeny that were assayed for the presence of ScPVs by RT-PCR and by extracting dsRNAs

ScPVs are resistant to the antiviral effects of Xrn1p
Prior studies have shown that pathways of RNA metabolism in yeasts are important for controlling the replication of totiviruses [9,[53][54][55][56][57][58]. Specifically, the 5' exoribonuclease Xrn1p cures dsRNA totiviruses when overexpressed, likely because of the degradation of uncapped viral RNAs. To determine if ScPVs are susceptible to attack by Xrn1p, a high copy plasmid encoding XRN1 was used to transform a strain of S. cerevisiae harboring totiviruses (L-A-lus and L-BC), an M2 satellite dsRNA, and ScPV3. After transformation, colonies were assayed for the loss of killer toxin production as an indication of Xrn1p restriction, as described previously [56]. The expression of XRN1 led to 50% of clones losing K2 killer toxin expression (S10 Fig), which was comparable to the loss of K1 expression (57%) [56]. Extraction of dsRNAs from non-killer yeasts revealed the expected loss of M2 in all strains, whereas killer yeast always maintained M2. Conversely, dsRNAs from ScPV3 and totiviruses were still observable, even after the loss of M2 (S10 Fig). This demonstrated that ScPV3 appears resistant to the overexpression of XRN1, which is effective at curing the L-A totivirus [56,57].

Discussion
This study reports the first detailed characterization of novel dsRNA viruses in S. cerevisiae in almost 50 years [12]. These viruses are from the family Partitiviridae, the first viruses of this type to be identified in yeasts of the Saccharomycotina subphylum [47,48]. ScPVs were predominantly associated with strains isolated from coffee and cacao beans from four genetically distinct yeast populations (South American and African cacao, South American and African coffee) that show evidence of admixture due to human migrations and farming practices [49]. ScPV1 and ScPV2 species were repeatedly found in yeasts isolated from coffee and cacao beans in different geographic locations, suggesting that human agricultural practices have distributed them with their host yeast strains [49]. ScPV3 is unique because it has only been identified in European S. cerevisiae strains associated with winemaking, which suggests either an independent lineage of ScPVs or outcrossing and invasion from strains associated with coffee and cacao.
Previous reports show that mycoviruses can be found in diverse species of fungi [59]. In S. cerevisiae, up to 22% of strains are thought to harbor mycoviruses due to the known association of viral infection with killer toxin production [46,60]. However, in coffee and cacao-

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae associated strains, 46% of strains harbor at least one dsRNA virus. Fermentation of coffee and cacao beans by different yeast and bacterial species, including genetically distinct strains of S. cerevisiae, is used to digest carbohydrates and proteins of the bean mucilage [61,62]. This could provide a unique environment for viral invasion of different yeast populations due to more frequent outcrossing and admixture. The prevalence of ScPVs in coffee and cacao strains could also be because they provide a specific advantage to S. cerevisiae during fermentation. As commercially available beans are the product of successful fermentation and have desirable flavor profiles, it would be interesting to survey failed fermentations to determine whether ScPVs are beneficial to coffee and cacao fermentations. Alternatively, it is also possible that coffee and cacao strains could be more susceptible to viral infection when compared to other yeast lineages, as genetic differences between strains and species of yeasts can render a strain more permissive to the replication of viruses and parasitic plasmids [9,56,58,[63][64][65][66].
ScPVs share a common ancestry with PVs of the Cryspovirus genus isolated from the protozoan pathogen C. parvum and other species of Cryptosporidium [67,68]. ScPVs have diverged from protozoan viruses as evinced by the low amino acid sequence identity of their RdRP (33-38%) and CP proteins (16-23%). These identities are close to the proposed cutoffs that would define ScPVs as a novel virus genus. Moreover, the smaller sizes of ScPV proteins set them apart from all previously described PVs. The genus name Cryspovirus was originally proposed to distinguish protozoan PVs from plant PVs [69]. The discovery of the ScPVs could justify reassessing the current taxonomic organization of the Partitiviridae.
The evolutionary relationship between ScPVs and CSpV1 suggests horizontal PV transfer between yeasts and protozoa. Cryptosporidium spp. have likely been associated with humans and livestock for millennia [70], and both can be chronically infected, shedding thousands of infectious oocysts per gram of feces. Similarly, yeasts are propagated in high numbers during fermentation. Together, this would release significant numbers of PV-containing cells into the environment that could have increased the likelihood of horizontal virus transfer. Although PVs lack genes that would enable mobilization, the extracellular transmission of viral particles has been demonstrated under laboratory conditions for both mycoviruses and protozoan viruses [71][72][73]. Previously, cross-kingdom virus transmission between fungi and plants has been shown to be possible, as inferred by phylogenetic discordance between virus and host, and has also been observed in the laboratory [74][75][76]. Additional efforts to characterize fungal and protozoan viruses will be vital in understanding the evolutionary origins of PVs and the frequency of viral transmission between kingdoms.
The CPs encoded by ScPVs are the smallest described in any member of the Partitiviridae with significant structural homology to other PV CPs, despite a lack of sequence homology. In other CP structures, the S-domain is larger because of extended loops between helices and the addition of P-domains that can interrupt the S-domain or are added as C-terminal extensions [26][27][28][29]. The predicted structure of ScPV CPs appears to most closely resemble the structure of the PCV1 CP but lacks an obvious P-domain in the S-domain [25]. The C-terminal domain of ScPV CP is 100 amino acids long and would be sufficient to form a P-domain, as they are �80 amino acids in length. The observed smooth particle morphology of ScPV1 would disfavor a prominent P-domain as seen with PCV1 and might be more similar to the "butte" domain of Fusarium poae virus 1 [29]. The ScPV C-terminus could play a role in virus particle assembly as in other PVs, but further empirical analysis will be needed to determine its role in particle assembly and stability.
The detailed study of host-virus interactions has been limited for PVs due to their infection of non-model organisms with a paucity of genetic tools. The similarity between ScPVs and CSpV1 would also enable a better understanding of PVs that infect a pathogenic protozoan without the need to collect oocysts from feces or the use of tissue culture models of C. parvum

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae infection. S. cerevisiae has been used for decades to study the replication of totiviruses and narnaviruses by using well-developed techniques that allow genetic crosses, genome manipulation, and high-throughput screening. This has included developing a reverse genetic system for launching narnaviruses from cloned cDNAs, the characterization of the antiviral systems of yeasts, and the ectopic expression of the totivirus Gag-Pol [9,[53][54][55][56][57][58]77,78]. However, the study of mycoviruses using S. cerevisiae as a model has been limited to those viruses that naturally infect S. cerevisiae, despite the successful laboratory infection with mycoviruses from filamentous fungi [79,80]. Therefore, the identification of ScPVs in S. cerevisiae and the discovery that they are transmitted during meiosis will allow the use of a myriad of genetic tools and strain collections to study host-virus interactions, antiviral mechanisms, and PV replication. Currently, it is unknown which cellular processes are required for efficient PV replication or how previously identified antiviral systems that restrict virus replication in yeasts interact with ScPVs [9,[53][54][55][56][57][58]. ScPVs appear resistant to Xrn1p, which suggests that these viruses employ mechanisms to protect their RNAs from degradation. This could include 5' end modification, cap-snatching, or secondary structure to block Xrn1p progression, as has been shown for other S. cerevisiae viruses [55,57,[81][82][83]. S. cerevisiae represents a unique opportunity to build on prior studies and further our understanding of the important and diverse members of the Partitiviridae.

Double-stranded RNA extraction
Double-stranded RNA was purified according to the rapid method of Crabtree et al. [45]. The yield was increased by recovering the supernatant from multiple phenol-chloroform extractions and loading it onto a single cellulose column. All strain information and their dsRNA content are compiled in S3 Table.

Sequencing of dsRNAs
Samples were prepared for sequencing by adding 3' poly(A) tails to purified dsRNAs and used as priming sites for cDNA synthesis, as previously described [45,46]. DNAs were prepared for Illumina sequencing using tagmentation [84], and sequencing of cDNAs was carried out according to Crabtree et al. [45]. For determining the sequence of the ends of PV genomes, the 5' RACE kit was used from Invitrogen as per the manufacturer's instructions. Completing the ScPV1 RdRP ORF required the following modifications to the manufacturer's protocol: Superscript IV reverse transcriptase (Invitrogen) was used instead of Superscript II. A longer GSP1 was designed to suit the higher temperatures of cDNA synthesis. And the 37˚C TdT incubation time was decreased to 2.5 min. Primers used to amplify the 5' ends of the segments of the various PVs are listed in S9 Table. 5' RACE products were cloned into the pCR8 vector using the TOPO-TA cloning kit (Thermo Fisher Scientific). All complete genome sequences of ScPVs have been deposited to NCBI Genbank under the accession numbers OP555746 (ScPV2-858 dsRNA2; CP), OP555747 (ScPV2-858 dsRNA1; RdRp), OP555748 (ScPV1-5509 dsRNA1; RdRP), OP555749 (ScPV1-5509 dsRNA2; CP), OP555750 (ScPV3-1172 dsRNA1; RdRP), and OP555751 (ScPV3-1172 dsRNA2; CP). All ScPV sequence accession numbers are listed in S10 Table. Sequencing analysis of dsRNA metagenomic data from S. cerevisiae 109 contigs (>300 nt in length) were identified as matching "Viruses (taxid: 10239)" using BLASTx (Max targets 10, Threshold 1 x 10 −6 ). Each hit was classified by virus family, and the coverage scores were totaled.

RNAse III digestions
Double-stranded RNAs were digested with ShortCut RNase III (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's protocol.

Total nucleic acid extraction from S. cerevisiae
Total nucleic acids were extracted from saturated liquid cultures of yeasts for RT-PCR as described previously [85].

Reverse transcription-PCR
Superscript IV (Invitrogen) was used for all RT-PCR reactions as directed by the manufacturer. Primers used to detect PVs are listed in S8 Table. Killer phenotype assays Killer toxin production by yeasts was measured using killer yeast agar plates (yeast extract, peptone, and dextrose (YPD) agar plates with 0.003% w/v methylene blue buffered at pH 4.6), as described previously [46].

Cycloheximide curing of satellite dsRNAs
10 μL of an overnight YPD culture of S. cerevisiae strain CYC1172 was transferred to YPD plates containing up to 1.5 mg/L of cycloheximide and incubated at ambient temperature for 3-5 days. Colonies growing on the highest concentration of cycloheximide were streaked to single colonies on standard YPD media and incubated at ambient temperature for 3-5 days. Ten colonies were assayed for the loss of the killer phenotype as described under "killer phenotype assays."

Phylogenetic analysis
RdRP protein sequences from ScPVs and other PVs from previously described genera (S7 Table) were aligned using MUSCLE and were inspected manually for accuracy. PROTTEST3 v3.4.2 was used to determine each dataset's appropriate amino acid substitution matrix [51]. The search space used consisted of all possible matrices and decorations with eight categories for the +I and +I+G models. The best-fit model was determined to be VT+I+G+F, with the next best model having a delta AIC of 28.52. PhyML v3.3.20220408 was used to create phylogenetic models [50]. PhyML parameters were chosen based on the results of PROTTEST3. The analysis consisted of 1000 bootstrap replicates, the VT amino acid substitution model was used, the proportion of invariable sites was determined by maximum likelihood, amino acid frequencies were estimated empirically by the frequency of occurrence in the dataset, the BEST tree search method was used for tree estimation, and the analysis was started with five random trees.

Virus particle purification
This protocol was adapted from that of Naitow et al. [86]. Cells were inoculated in 1000 mL yeast peptone dextrose (YPD) liquid media and incubated at 30˚C for 24 hours. Cells were harvested by centrifugation for 5 min at 3800 × g and washed with double-deionized water. Washed cells were suspended in spheroplasting buffer (100 mM Tris-HCl, 1M D-sorbitol, 20 mM β-mercaptoethanol, 0.5 mg/mL Zymolyase 20T, pH 7.6) and incubated for 90 min at ambient temperature, stirring gently. Digested cells were harvested by centrifugation for 5 min

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae at 3,800 × g and suspended in buffer A (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, pH 7.6), after which they were lysed by passing the cell suspension twice through a French pressure cell press (Thermo Scientific) at 20,000 psi. Cell debris was removed by centrifugation for 20 min at 9,600 × g, and the supernatant was adjusted to 3% PEG-8000 and 0.5 M NaCl before rocking gently for 60 min at 4˚C. The precipitate was harvested by centrifugation for 20 min at 9,600 × g, gently suspended in 15 mL of 50 mM sodium phosphate buffer (pH 7.0), and incubated on ice overnight with rocking. Insoluble precipitates were removed by centrifugation at 7,000 × g for 10 min. The supernatant was layered onto 5 mL 20% sucrose cushion in an ultracentrifuge tube (Thermo Scientific, Nalgene High-Speed Round-Bottom PPCO Centrifuge Tubes) and centrifuged at 80,000 × g for 2 hours at 4˚C. The pellet was recovered and suspended in 500 μL sodium phosphate buffer and layered onto a discontinuous sucrose gradient (10-50% in 5% increments of 1 mL each). This was centrifuged at 70,000 × g for 2 hours at 4˚C.

Transmission electron microscopy
For the negative staining of VLPs, 5 μl of purified particles from strain YO1126 were applied to a 400-mesh copper grid coated with carbon-Formvar (Ted Pella) for 20 seconds, wicking with a filter paper, and washed three times on 50 μl water droplets. A staining solution of 2% uranyl acetate (pH � 3) was added to the sample-side of the grid, wicking, and reapplied for 20 seconds before removal by wicking and lateral aspiration. Images were obtained on an FEI Tecnai F20 transmission electron microscope, captured with a BM UltraScan camera, and stored in digital micrograph 3 format.

Molecular modeling
Collabfold version 1.4 was used to generate models using the open reading frame of the CP and RdRP of viruses CSpV1, ScPV1-5509, ScPV2-858, ScPV3-1172 with the following parameters (msa_mode: MMseqs2 (uniref+environment); model_type: auto; pair_mode: unpaired+paired; num_recyles: 3). Five unrelaxed structures of each CP and RdRP were generated. RdRP models were generated with similar local distance difference test (pLDDT) values per residue (S4 Fig). The highest average pLDDT score for each model was chosen for energy minimization and analysis. Energy minimization was carried out for each model using standard energy minimization protocol described in our previous study [17]. A dodecahedron box was generated around each model and solvated using a 10 Å layer of TIP3P. Ions were added to maintain charge neutrality at a concentration of 0.15 mol/L using Na + and Clions. The force field parameter used for protein and ions was AMBER99SB*-ILDNP. Each system underwent energy minimization using the steepest descent algorithm for 10,000 steps via the GROMACS package. Stereochemical analysis was carried out with the SWISS-MODEL structure assessment tool for each model after the energy minimization (https://swissmodel.expasy.org/) to verify that model sterics were physically reasonable after energy minimization. All PDB formatted files for each relaxed model can be found in S1 File. The final energy minimized RdRP and CP model structures were submitted to the DALI server to identify structural homologs (DALI data presented in S2 File). The cealign command in the PyMOL visualization software package was used to identify regions of homology between CPs and homologous CP structures identified by DALI [87].

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae leucine, lysine, tryptophan, methionine, arginine), 0.004% (adenine, uracil, tyrosine), 0.01% phenylalanine, 0.035% threonine, filter-sterilized). The sporulation rate was monitored by microscopy. When the sporulation rate was at least 25%, 50 μL of cell suspension was centrifuged, and the supernatant was removed. Cells were suspended in 50 μL 1 mg/mL yeast lytic enzyme (A. G. Scientific) and digested for precisely 12 min, after which the reaction was quenched by adding 0.5-1 mL sterile water. Asci were dissected with a 25 μm fiber optic cable glued to a micromanipulator mounted onto a Nikon Eclipse 50i microscope. Ascospores were germinated at 30˚C, grown to single colonies, and used for additional analysis.

Transmission of ScPVs
Stable haploids of strains YO1126 and CYC1172 were obtained by HO disruption and sporulation to create the diploid strains YTag006 and Ytag015, respectively. Primers NTT033 and NTT034 were used to amplify the ho::HygMX cassette from strain YJM975 MATα ho::HygMX, using purified chromosomal DNA as a template. 1 μg of the amplicon was used to transform YO1126 and CYC1172. After selection on hygromycin (0.3 mg/mL), PCR verification was performed by probing HO with the same primers. HO/ho::HygMX diploid cells were sporulated and dissected. The dissection plate was replica-plated onto YPD containing hygromycin. Mating type was determined by PCR, using primers NTT041, NTT042, and NTT043. This procedure created the stable haploid strains YTag009 and YTag058 from YTag006 and Ytag015, respectively. YTag009 and YTag058 were each crossed with BY4741 using a method from the Meneghini laboratory (personal communication). Briefly, YTag009 was mixed with BY4741 in a ratio of � 1:100 and spotted onto YPD agar and incubated at 30˚C overnight. Cells were streaked to single colonies on YPD plates on which had been spread 125 μL 1 mg/mL alpha factor pheromone and incubated at 30˚C for 1-2 days; this process was repeated once more. Diploids were verified by PCR, using primers NTT041, NTT042, and NTT043. This method created YTag121 and YTag085. ScPV infection was verified by RT-PCR and dsRNA extraction followed by agarose gel electrophoresis.

ScPV stability
Yeast strains were streaked onto two YPD agar plates each and incubated at either 25˚C or 4˚C for 6 weeks. Cells were cultured in 12 mL YPD broth and incubated at 25˚C for 2 days, and their dsRNAs were extracted and analyzed by agarose gel electrophoresis.

Halo mating type assays
Strains DBY7730 and DBY7442 were streaked onto YPD agar and incubated at 30˚C for 1-2 days. One colony from each was used to inoculate overnight cultures in YPD broth, shaken at 30˚C. The cell suspensions were diluted 1:20, and 400 μL of each was spread onto a 100 mm YPD agar plate and incubated at 30˚C for 30 min. Strains of interest were pinned onto the lawn using sterile toothpicks, and the plates were incubated overnight at 30˚C. A zone of growth inhibition surrounding a colony indicated that it is the opposite mating type of the lawn strain.

Curing of viruses and dsRNA satellites from S. cerevisiae by XRN1 expression
The strain S. cerevisiae YTag085 (an M2 killer yeast) was transformed with the high copy plasmid pPAR219 to express XRN1 cloned from S. cerevisiae [56]. The loss of the killer phenotype in 183 clones was confirmed by a killer assay using two different K2-susceptible strains of yeast

PLOS PATHOGENS
Viruses of the Partitiviridae in Saccharomyces cerevisiae (BY4741 and DBY7730). Cured clones were then assayed for dsRNAs by cellulose chromatography.

Strains of yeast
All strains of yeast used in this manuscript are listed in S11 Table. Supporting information S1 Fig. Contig size and